Evaluation of three protocols for direct susceptibility testing for gram negative-Enterobacteriaceae from patient samples in Uganda with SMS reporting

In Uganda, the challenge of generating and timely reporting essential antimicrobial resistance (AMR) data has led to overreliance on empirical antibiotic therapy, exacerbating the AMR crisis. To address this issue, this study aimed to adapt a one-step AMR testing protocol alongside an SMS (Short Message Service) result relay system (SRRS), with the potential to reduce the turnaround time for AMR testing and result communication from 4 days or more to 1 day in Ugandan clinical microbiology laboratories. Out of the 377 samples examined, 54 isolates were obtained. Notably, E. coli (61%) and K. pneumoniae (33%) were the most frequently identified, majority testing positive for ESBL. Evaluation of three AMR testing protocols revealed varying sensitivity and specificity, with Protocol A (ChromID ESBL-based) demonstrating high sensitivity (100%) but no calculable specificity, Protocol B (ceftazidime-based) showing high sensitivity (100%) and relatively low specificity (7.1%), and Protocol C (cefotaxime-based) exhibiting high sensitivity (97.8%) but no calculable specificity. ESBL positivity strongly correlated with resistance to specific antibiotics, including cefotaxime, ampicillin, and aztreonam (100%), cefuroxime (96%), ceftriaxone (93%), and trimethoprim sulfamethoxazole (87%). The potential of integrating an SRRS underscored the crucial role this could have in enabling efficient healthcare communication in AMR management. This study underscores the substantial potential of the tested protocols for accurately detecting ESBL production in clinical samples, potentially, providing a critical foundation for predicting and reporting AMR patterns. Although considerations related to specificity warrant careful assessment before widespread clinical adoption.


Isolate identification and ESBL status
A total of 54 isolates were obtained from 45 out of the 377 samples.Among these, the most frequently identified isolates were E. coli 33 (61%) and K. pneumoniae 18 (33%).Additionally, Citrobacter spp and Enterobacter spp 3(6%) were also present.Notably, the majority of the isolates tested positive for ESBL.The 33 isolates that met the laboratory's SOPs for reporting during the study all tested positive for ESBL, indicating 100% ESBL prevalence (Table 1).

Accuracy of AMR testing protocols
Protocol A The test had a high sensitivity (100% of true positives were detected), but a very low specificity (no true negatives were detected).The positive predictive value (PPV) was 75.4% while the negative predictive value (NPV) could not be calculated.
Protocol B The test had a high sensitivity (100% of true positives were detected), it had a relatively low specificity (only 7.1% of true negatives were detected).The PPV was 77.2% while the NPV was 100%.
Protocol C The test had a high sensitivity (97.8% of true positives were detected), it had a very low specificity (no true negatives were detected).The PPV was 75.9% while the NPV could not be calculated (Table 2).

SMS result relay system (SRRS) integration
To enhance communication within the A-LIS framework, an SRRS was integrated using the Africa's Talking platform.This involved seamlessly integrating the SRRS into A-LIS via the Africa's Talking, necessitating users to have accounts with this third-party provider.Additionally, a unique Application Programming Interface (API) key was generated to facilitate SMS transmission through the integrated system.

SMS service provider (SP) selection
The selection of an SMS SP was determined by evaluating the cost structure, categorized into two bulk SMS and sender identification configuration.After careful consideration, SMS SP2 emerged as the optimal choice due to its competitive pricing and absence of monthly maintenance fees (Table 4).

Architecture and design
The architecture and design of the SRRS were developed for simplicity and efficiency in mind.The user experience includes the following steps: User login: Users are required to log in to A-LIS; sample selection: After logging in, users can access samples and search for specific requiring SMS notifications; sending SMS: users can initiate the SMS sending process by clicking the "Send SMS" button, providing valid phone numbers and composing message.The system automatically delivers messages to recipients (Fig. 1).

Discussion
The findings of this study depict a troubling scenario of widespread multidrug-resistant (MDR) ESBL-PE, aligning with similar reports from Uganda and other global regions [15][16][17][18]29   www.nature.com/scientificreports/ andaztreonam, even extending to ceftriaxone, often considered a dependable last-resort option [15][16][17][18]29 .This pervasive resistance presents a substantial challenge for clinicians, constraining their choices for empiric therapy and elevating the risk of treatment failure [15][16][17][18][29][30][31] .This situation underscores the dire urgency emphasized by the World Health Organization (WHO), which classifies ESBL-PE as a high priority threat 32 .Their ability to render even broad-spectrum antibiotics ineffective translates into longer hospital stays, increased healthcare costs, and unfortunately, higher mortality rates [21][22][23] .The pervasiveness of these organisms in this study underscores the urgency of accurate and timely detection, not just for patient well-being but also for the sustainability of healthcare systems grappling with the rising tide of AMR.
More importantly, the pervasiveness of ESBL-PE offers a valuable opportunity.Their presence can serve as a potent proxy marker, potentially providing early warning signs of broader resistance patterns in other bacterial pathogens within the same setting 33 .This additional layer of insight can significantly bolster surveillance efforts and inform proactive interventions to safeguard the efficacy of antibiotics before resistance spreads further.
While the overarching situation seems distressingly similar worldwide, a closer examination of regional variations, including antibiotic prescribing patterns, healthcare infrastructure, and patient demographics, could unveil unique contributing factors 34,35 .Recognizing these local nuances is imperative for tailoring interventions and effectively combating this shared threat.In light of this disconcerting picture, clinicians must embrace antibiotic stewardship practices, prioritizing therapy whenever feasible and customizing regimens based on local resistance patterns 36,37 .Furthermore, implementing robust infection control measures is essential to prevent nosocomial transmission of these MDR ESBL-PE 38,39 .Accurate and timely detection of ESBL-PE is crucial for effective antibiotic therapy and preventing resistance spread 40,41 .While the established gold standard protocol remains reliable, its limitations, such as longer turnaround times and higher costs 42 , highlight the need for alternative approaches.This study compared three distinct AMR testing protocols against the gold standard, assessing their capacity to reliably detect ESBL production.
The findings revealed clear strengths and weaknesses among the protocols.Protocols A and C boasted impressive sensitivity, accurately identifying nearly all ESBL-PE isolates.However, their extremely low specificity could lead to overdiagnosis, that could potentially misguide antibiotic therapy and increase unnecessary antibiotic use 37,43 .Conversely, Protocol B exhibited perfect sensitivity but suffered from lower specificity, raising concerns about missed cases.This underlines the critical need for a balanced approach that prioritizes both accurate detection (sensitivity) and avoiding false positives (specificity).
Moving beyond raw numbers, the study also considered the positive predictive values (PPV) of each protocol.Positive predictive values tell the likelihood that a positive test result truly reflects the presence of ESBL-PE 44 .Notably, the analysis revealed that ESBL positivity is a strong predictor for resistance to several key antibiotics, including ampicillin, cefuroxime, ceftriaxone, ceftazidime, cefotaxime, aztreonam, and trimethoprim sulfamethoxazole.This emphasizes the clinical significance of accurate ESBL detection in tailoring antibiotic regimens and ensuring effective treatment, reducing the risk of resistance development and treatment failure 19,20,45,46 .
These findings have immediate implications for clinical practice.While Protocol A and C offer the advantage of high sensitivity, their low specificity necessitates further refinement or combination with confirmatory tests to avoid misdiagnosis and overuse of antibiotics.Conversely, Protocol B, despite its lower sensitivity, might be suitable for settings where rapid diagnosis is crucial and over-treatment with broad-spectrum antibiotics poses a lesser risk.Ultimately, the choice of an optimal protocol should consider the specific clinical context, resource limitations, and desired balance between sensitivity and specificity.
By acknowledging the limitations of existing protocols and exploring the clinical implications of their performance and PPV, this study paves the way for optimizing accurate and timely ESBL detection strategies.Beyond comparing AMR testing protocols, this study investigated the potential of an SRRS to enhance communication within the A-LIS framework.An API integration with Africa's Talking platform was designed to enable automated SMS notification of test results to healthcare providers.These rapid notifications hold promise for improving patient care and combating AMR by empowering clinicians to make informed decisions 47,48 .Clinicians can prescribe targeted antibiotics based on individual AMR profiles, potentially initiating treatment faster and reducing unnecessary tests.Moreover, the potential for these improved clinical outcomes suggests this system could have additional, long-term economic benefits by promoting judicious antibiotic stewardship and mitigating associated healthcare costs.The study assessed the cost implications of using various SMS plans and sender configurations with different SMS SPs in Uganda, identifying SMS SP2 as a potentially cost-effective option due to its competitive pricing and lack of monthly fees.
However, it is important to note that this cost analysis was limited to one country (Uganda) and did not involve actual implementation of the SRRS in a clinical setting.Therefore, the potential impact on institutional and patient costs, as well as patient experiences, remains to be investigated.Similarly, the effectiveness of the SRRS in addressing communication barriers and influencing turnaround times requires further evaluation through real-world implementation studies.
While the focus of pilot implementation studies would naturally be on cost-effectiveness and user experience, a comprehensive evaluation necessitates consideration of the system's broader impact.This includes assessing its clinical effectiveness by evaluating the influence of rapid test results on antibiotic prescribing and patient outcomes.Additionally, acknowledging and addressing potential technical challenges such as data security, system integration, and user adoption is crucial for a holistic picture of the system's feasibility and potential success.
Moving forward, research efforts should focus on assessing the feasibility and impact of integrating both the rapid AMR testing protocols and the SRRS within operational healthcare settings.This would involve evaluating not only the clinical effectiveness, cost-effectiveness, and user experience of such a system, but also addressing potential technical challenges associated with its implementation.By bridging the communication gap between laboratories and clinicians, innovative approaches like SRRS hold promise for improving patient care and combating the growing threat of AMR.However, rigorous research and pilot implementation studies are crucial to validate the real-world effectiveness of this approach and pave the way for its successful translation into clinical practice.
Vol:.( 1234567890 While this study provides valuable insights into alternative AMR testing protocols and the integration of an SRRS in a resource-limited setting, several limitations warrant consideration when interpreting its generalizability.The relatively small sample size of 54 isolates highlights the need for further research with larger, more diverse populations to solidify the protocols' performance and scalability across varied patient demographics and resistance patterns.Additionally, generalizability is restricted by the study's single-setting environment in Uganda, where healthcare infrastructure and resource availability may differ significantly from other regions and countries.The focus on accurate and timely ESBL detection, while clinically significant, represents only one facet of the complex AMR landscape, demanding future exploration of the protocols' efficacy against other prevalent mechanisms crucial for informed antibiotic prescribing.Furthermore, the feasibility and cost-effectiveness of the SRRS integration, relying on Africa's Talking and SMS SP2, may not translate directly to other settings with varied third-party providers, telecommunications infrastructures, and regulatory landscapes.Resource constraints also limited the study's scope, precluding analysis of additional AMR mechanisms and antibiotic classes as well as a deeper dive into cost-effectiveness across diverse settings.Finally, external validation in different clinical settings and regions is crucial to confirm the findings' generalizability and ensure their successful translation into diverse healthcare systems, beyond the specific conditions of this study.By acknowledging and addressing these limitations, the study paves the way for further research aimed at validating and optimizing these interventions within broader contexts, ultimately driving their impactful implementation in the global fight against AMR.

Conclusions
This study provides valuable insights into the potential of three alternative AMR testing protocols and an SRRS for resource-limited settings.While sensitivity for ESBL-PE detection was promising, further investigation is needed to optimize specificity before clinical implementation.The study also highlights the significant role of efficient communication in AMR management, with the SRRS demonstrating potential for improved patient care.Moving forward, research efforts should focus on expanding the sample size and diversifying study settings to solidify the external validity of these findings.Additionally, investigations into the long-term sustainability, user experience, and clinical outcomes associated with the integrated SRRS are crucial to assess its real-world feasibility and impact.By building upon this study's foundation, future research can pave the way for optimizing these interventions and contributing significantly to the fight against AMR.

Study design and setting
This was a cross-sectional study conducted at the clinical microbiology laboratory (CML) in the Department of Medical Microbiology at Makerere University College of Health Sciences (MakCHS).The CML was accredited by the College of American Pathologists in 2018 and has a laboratory information management system, A-LIS.The CML routinely receives and tests for AMR in clinical samples from mainly MNRH.Mulago National Referral Hospital was founded in 1913 and is a teaching hospital for MakCHS 49 .It also serves as a general hospital for the Kampala metropolitan area 49 .This study was conducted between February and June 2023.

Study population and sampling
In this study, we utilized used part of clinical samples initially received by the CML for routine AMR testing.Only 377 samples-including urine (299), blood (24), pus swabs (16), pleural fluid (8), tracheal aspirates (4), and sputum (26)-were accessible for analysis in this study.Among these, 45 samples exhibited growth suitable for following up based on protocols (a-b).Any samples lacking growth for follow-up based on the protocols (a-b) were excluded.

Culture and AST (Gold standard)
At CML, clinical samples underwent a standardized process over approximately four days upon delivery.The first step involved culturing the samples to isolate and identify pathogenic bacteria.Culturing utilized MacConkey agar, known as a "general-purpose selective agar" or "non-antibiotic containing selective agar" 50 .The isolation process took up to 24 h (1 day).Once isolation was complete, various methods were employed, including Gram staining and biochemical tests (Triple Sugar Iron, Simmon's Citrate, Urease, and Sulphur Indole and Motility), to identify the resulting growth 51,52 , which also took up to 24 h (1 day).In cases requiring purification, the process extended beyond 24 h.Following identification, Kirby Bauer disk diffusion AST was conducted.This method exposed the bacteria to specific antibiotic concentrations, observing growth or inhibition.Interpretation relied on established criteria for each bacteria-antibiotic combination following CML SOPs aligned with Clinical Laboratory Standards Institute Guidelines (CLSI) (Version 2021).AMR testing itself took up to 24 h (1 day).Subsequently, the laboratory reported AMR testing results to healthcare facilities or clinicians in PDF format, including patient information, identified pathogens, tested antibiotics, and susceptibility results.These PDF reports were generated through A-LIS and then printed.The overall process typically took around four days to determine bacterial sensitivity or resistance to antibiotics.Despite being within the recommended turnaround times, variations in result delivery to clients occasionally extended this timeframe.

Culture and AST (Protocols A, B, and C)
This study implemented three potential one-step AMR testing protocols.These protocols, labeled as Protocol A, Protocol B, and Protocol C, were based on the following agar media: ChromID ESBL (bioMe´rieux), MacConkey agar containing 16 µg/ml of ceftazidime, and MacConkey agar containing 4 µg/ml of cefotaxime, respectively.ChromID ESBL served as an agar designed for isolating and detecting ESBL production.It featured a rich nutrient capacity and an antibiotic, cefpodoxime, known for inhibiting the growth of Gram-positive bacteria and yeast 53 .

Figure 1 .
Figure 1.Sequencial flow within the SRRS: Firstly, (a) showcases the user login interface, detailing the initial step for users accessing the system.Following successful login, the interface is depicted in (b), showcasing the system interface after user authentication.Subsequently, (c) exhibits the sample selection interface, enabling users to access and search for specific samples necessitating SMS notifications.The process of sending SMS notifications initiates with (d), where users input recipient phone numbers and compose messages.Continuing from figure (d), (e) provides further insight into the user interface for sending SMS notifications.Lastly, (f) illustrates the interface displaying received SMS notifications on a mobile device. https://doi.org/10.1038/s41598-024-53230-wwww.nature.com/scientificreports/

Table 1 .
Isolate identification and ESBL status by resistance testing protocol.
. Much like other studies conducted elsewhere, this study highlights remarkably high resistance to crucial cephalosporins such as ampicillin, cefotaxime,

Table 2 .
Accuracy of AMR testing protocols based on the analysis of the 45 samples.PPV Positive predictive value; NPV Negative predictive value; NC Not calculable due to the absence of true negatives; CI Confidence interval.

Table 3 .
PPVs of ESBL positivity for AMR in isolates.

Table 4 .
Bulk SMS plans and sender identification configurations per SMS SP.USD United States Dollars; SMS SP1 SMS Service provider 1; SMS SP2 SMS Service provider 2.